Nitric oxide synthase (NOS) regulates nitric oxide (NO) synthesis and thereby its dual biological activities as a diffusible messenger for platelet aggregation, blood pressure regulation, neurotransmission, long-term potentiation, and also as a cytotoxic agent for defense against tumor cells and parasites. Three NOS enzymes, the inducible (iNOS), endothelial (eNOS), and neuronal (nNOS) isoforms, achieve their key biological functions via intriguing regulations of their electron transfer mechanism and an assembly of six cofactors. Each subunit of the NOS dimer has two modules joined by a calmodulin-binding linker: an oxygenase module (NOSox) with heme, tetrahydrobiopterin (H4B), Zn ion, and Arginine binding sites forming the catalytic center for NO production, and a reductase module (NOSred) with NADPH, FAD, and FMN sites supplying electrons to the heme. Our overall goal is to characterize the detailed structural biochemistry underlying the active site interactions, catalysis, isozyme-specificity, assembly, regulation, and both inter-domain and inter-protein interactions of NOS enzymes. Our characterizations of the independently functional dimeric NOSox and NOSred modules, and of calmodulin (CaM) bound to the CaM-binding peptide, provide a powerful framework for interpreting NOS structural biochemistry. Our progress to date prompts four proposed Aims, which are driven by specific hypotheses. We now propose integrated structural, mutational and biophysical experiments to test these hypotheses and to address specific critical and challenging unanswered questions. How do NOSox, NOSred and CaM assemble for function? What is the mechanism for rate-limiting electron transfer from the NOSred FMN to the NOSox heme? How do isozyme-specific features tune and regulate NOS activity? How is NOS activity regulated through interactions with its protein partners? Our interdisciplinary experiments on NOS domains and full-length proteins will characterize active-site interactions, key assemblies, conformational switching mechanisms, and inter-protein interactions. We expect to characterize prototypical sets of structures and mutant enzymes, functional complexes with inhibitors and with protein partners, and to define the structural chemistry underlying the exquisite regulation of NO( synthesis. Deuterium Hydrogen Exchange Mass Spectrometry (DXMS) and advanced Small-Angle X- ray Scattering (SAXS) combined with computationally-aided design of mutants to lock, strengthen or block interactions (including designed disulfide linkages) will test and complement high resolution crystallographic structures. The expected outcome of the proposed research is a detailed molecular understanding of the activity, inhibition, and regulation of NOS isozymes relevant to important aspects of their biology and medical importance for blood pressure regulation, stroke, septic shock, cancer and inflammatory damage.